The field of the invention is drug delivery, including nucleic acid delivery.
Delivery of one or more types of biologically active molecule to an animal or to an animal tissue forms the basis of modem pharmacology. In order to achieve the fullest therapeutic or prophylactic effect, the composition and method used to deliver the bioactive agent must provide the proper amount of the agent to the appropriate tissue(s) of the animal, in an active or activatable form, at an appropriate point in time, and for a sufficient duration. Despite thousands of years of pharmacological research and practice, there remains a critical need for compositions and methods of delivering polyionic bioactive agents, particularly in a sustained-release manner.
Numerous polyionic bioactive agents are known in the art, including both polyanionic and polycationic bioactive agents. Nucleic acids, in particular, have proven difficult to deliver effectively to the animal and in a sustained-release manner. The difficulties in delivering nucleic acids have persisted despite the intense and increasing desire of researchers and clinicians to be able to deliver nucleic acids to animal tissues, particularly to human tissues. In December, 1995, the U.S. National Institutes of Health issued a xe2x80x9cReport and Recommendations of the Panel to Assess the NIH Investment in Research on Gene Therapy (Orkin et al., 1995, National Institutes of Health, Bethesda, Md.). In this Report, it was recognized that the development of gene therapy approaches to disease treatment was being inhibited, in part, by a dearth of effective gene transfer vectors. The Report recognized a need for further research applied to improving vectors for gene delivery.
Among the physiological phenomena which can inhibit administration of a nucleic acid to an animal tissue are the following.
Inability to direct the nucleic acid to cells of the desired tissue.
Inability of the nucleic acid to cross membranes of cells of the desired tissue.
Nucleolytic digestion of the nucleic acid prior to its delivery to cells of the desired tissue.
Nucleolytic digestion of the nucleic acid within cells of the desired tissue prior to transfer of the nucleic acid to a location within the cells at which the nucleic acid may exert its intended effect.
Clearance of the nucleic acid from the animal""s system before the nucleic acid has been delivered to a sufficient fraction of cells of the desired tissue.
Inability to achieve an adequate dosage of the nucleic acid at the desired tissue.
A desirable nucleic acid vector will permit administration that is not significantly inhibited by these phenomena.
Numerous compositions and methods are known for delivering a nucleic acid to an animal tissue. Such compositions include xe2x80x9cnakedxe2x80x9d (i.e. non-complexed) nucleic acids, nucleic acids complexed with cationic molecules such as polylysine and liposome-forming lipids, and virus vectors.
Naked nucleic acids can be taken up by various animal cells, but are subject to nucleolysis, both inside and outside of cells that take them up. For example, it is known that cells in wounded tissue (e.g. cells lining an incision made in a tissue) are particularly amenable to taking up naked nucleic acids. Examples of such cells include, but are not limited to, fibroblasts, capillary endothelial cells, capillary pericytes, mononuclear inflammatory cells, segmented inflammatory cells, and granulation tissue cells.
The use of nucleic acid analogs which are relatively resistant to nucleolysis is known. Such analogs include, for example, phosphorothioate nucleic acid analogs. However, in some situations, particularly where incorporation of the nucleic acid into the genome of the target cell is desired, the use of nucleic acid analogs can be undesirable. Targeting of naked nucleic acid vectors to particular animal tissues can be difficult, particularly in situations in which the tissue is normally bathed by a liquid in which the vector may be carried away from the tissue site.
Compositions for sustained release of naked nucleic acids are known, but such compositions have many of the same drawbacks of other naked nucleic acid vectors, namely, that the nucleic acids released from the compositions may not be efficiently taken up by cells of the desired tissue and that the nucleic acids released from the compositions are susceptible to nucleolysis. Examples of such compositions include compositions comprising naked nucleic acids in a biodegradable polymer matrix. Another shortcoming of such compositions is that they can be difficult to target to specific tissues in order to achieve localized delivery of the nucleic acid. Such compositions generally occur in liquid form, which must be injected at the desired site, but is capable of flowing from the site of administration to other sites.
Numerous vectors comprising a nucleic acid complexed with a compound to improve stability or uptake of the nucleic acid by a target cell have been described. Such compounds include, by way of example, calcium phosphate, polycations such as diethylaminoethyl-dextran, polylysine, or polybrene, and liposome-forming lipids such as didocylmethylammonium bromide and Lipofectamine(trademark). Many of these compounds are toxic, or produce undesired reactions, when administered to patients. Thus, while nucleic acid vectors comprising a nucleic acid complexed with one of these compounds may be useful for transfection of cultured cells, these vectors are not useful for delivering nucleic acids to cells in an animal tissue.
Virus vectors are generally regarded as the most efficient nucleic acid vectors. Recombinant replication-defective virus vectors have been used to transduce (i.e., infect) animal cells both in vitro and in vivo. Such vectors have included retrovirus, adenovirus, adeno-associated virus vectors, and herpes virus vectors. While highly efficient for gene transfer, a major disadvantage associated with the use of virus vectors is the inability of many virus vectors to infect non-dividing cells. Another serious problem associated with the use of virus gene vectors is the potential for such vectors to induce an immune response in a patient to whom they are administered. Such an immune response limits the effectiveness of the virus vector, since the patient""s immune system rapidly clears the vector upon repeated or sustained administration of the vector. Furthermore, insertion of a gene into the genome of a cell by a virus vector may induce undesirable mutations in the cell. Other problems associated with virus gene vectors include inability to appropriately regulate gene expression over time in transfected cells, potential production and transmission to other humans of harmful virus particles, local and general toxicity, undesirable immunogenicity, and unintended disruption of target or other cell metabolism.
What is needed are compositions and methods which can be used to deliver nucleic acids to cells of a desired tissue in an animal in a form in which the nucleic acid can cross the cell membranes in a relatively nuclease-resistant form, preferably for a period sufficient to permit the nucleic acid to enter a substantial fraction of the cells of the desired tissue. Also preferably, the nucleic acid vector should not induce toxicity or other significantly harmful reactions in the animal.
Proteins are another class of polyionic bioactive agents which have proven to be difficult to administer to animal tissues in many cases. Proteins, often being very large molecules, are difficult to transfer across a cell membrane. In addition, certain proteins, including integral membrane proteins such as certain membrane-bound receptors, must be inserted into the cell membrane in the correct orientation in order to exhibit their characteristic biological activity. Furthermore, proteins are susceptible to degradation by the action of proteolytic enzymes, which are common in biological systems. Proteins may be delivered to a tissue as the intact protein, as subunits which assemble at the site of delivery, as propeptides which are proteolytically cleaved at the site of delivery to yield active protein or protein subunits, or in the form of one or more nucleic acids which encode the protein or its constituent subunits. Delivery of nucleic acids encoding the protein or its subunits has the advantages that the nucleic acids may be easier to transfer across the cell membrane, that they are not subject to proteolysis, and that expression of the protein from the nucleic acids may result in properly oriented, active protein. A significant need exists for compositions and methods of delivering a protein to a tissue of an animal.
The compositions and methods of the present invention satisfy the needs identified above.
The invention relates to a composition for delivery of a polyionic bioactive agent. The composition comprising the polyionic bioactive agent and a matrix having an exterior portion. At least most of the polyionic bioactive agent present at the exterior portion of the matrix is in a condensed form. In one embodiment, substantially all of the polyionic bioactive agent present at the exterior portion of the matrix is in a condensed form. In another embodiment, the exterior portion has an exterior surface, and the polyionic bioactive agent is present substantially only on the exterior surface of the exterior portion of the matrix. The matrix may, of course, comprise a plurality of the exterior portions.
In one aspect of the composition of the invention, the matrix further comprises an interior portion having the polyionic bioactive agent suspended therein, and less than most of the polyionic bioactive agent suspended in the interior portion of the matrix is not in a condensed form. In this aspect, the matrix may comprise a plurality of alternating the exterior portions and the interior portions.
In another aspect of the composition of the invention, the exterior portion comprises a polyionic condensing agent having a charge opposite that of the polyionic bioactive agent. Preferably, the polyionic bioactive agent is a polyanionic bioactive agent, and the polyionic condensing agent is a polycationic condensing agent, such as one selected from the group consisting of a polylysine, polyarginine, polyornithine, polyhistidine, myelin basic protein, a low molecular weight glycopeptide, a cationic amphiphilic alpha-helical oligopeptide having a repeating sequence, a galactosylated histone, Mg2+, Ca2+, Co3+, La3+, Al3+, Ba2+, Cs+, polybrene, spermine, spermidine, prolamine, polyethylenimine, putrescine, cadaverine, and hexamine. Preferably, the polycationic condensing agent is poly-L-lysine.
Exemplary polyanionic bioactive agents include, but are not limited to, a nucleic acid, a nucleic acid analog, a plasmid, a linear DNA molecule, a linear RNA molecule, an antisense oligonucleotide, an expression vector, a transformation vector, a transfection vector, a ribozyme, a transcribable vector comprising a DNA molecule encoding a ribozyme, a viral fragment, a cosmid, a DNA molecule encoding a portion of the genome of an organism, a cDNA molecule, a gene fragment, a single-stranded DNA molecule, a double stranded DNA molecule, a supercoiled DNA molecule, a triple-helical DNA molecule, and a Z-DNA molecule.
In certain embodiments, the polyanionic bioactive agent is selected from the group consisting of an expression vector encoding a wound healing therapeutic protein, an expression vector encoding an anti-restenotic protein, and an anti-restenotic antisense oligonucleotide. The wound healing therapeutic protein may, for example, be selected from the group consisting of TGF-xcex2, FGF, PDGF, IGF, M-CGF, BMP, GH, and PTH. The anti-restenotic protein may, for example, be selected from the group consisting of TPA, TGF-xcex2, FGF, Rb, p21, and TK. The anti-restenotic antisense oligonucleotide may, for example, be selected from the group consisting of a c-myb antisense oligonucleotide, a c-myc antisense oligonucleotide, and a PCNA antisense oligonucleotide.
In one embodiment of the composition of the invention, the polyionic bioactive agent is a polycationic bioactive agent, and the polyionic condensing agent is a polyanionic condensing agent. The polyanionic condensing agent may, for example, be a nucleic acid. The polycationic bioactive agent may, for example, be selected from the group consisting of a DNA-binding protein, a histone, a cationic protein, a polyamine, cadaverine, putrescine, spermidine, and spermine.
In another embodiment of the composition of the invention, the matrix is selected from the group consisting of a charged biocompatible material, a biocompatible polymer, a biodegradable polymer, a biocompatible biodegradable polymer, polylactic acid, polyglycolic acid, polycaprolactone, a copolymer of polylactic acid and polyglycolic acid, a copolymer of polylactic acid and polycaprolactone, a copolymer of polyglycolic acid and polycaprolactone, a polygylcolide, a polyanhydride, a polyacrylate, a polyalkyl cyanoacrylate, n-butyl cyanoacrylate, isopropyl cyanoacrylate, a polyacrylamide, a polyorthoester, a polyphosphazene, a polypeptide, a polyurethane, a polystyrene, a polystyrene sulfonic acid, a polystyrene carboxylic acid, a polyalkylene oxide, a polyethylene, a polyvinyl chloride, a polyamide, a nylon, a polyester, a rayon, a polypropylene, a polyacrylonitrile, an acrylic, a polyisoprene, a polybutadiene, a polybutadiene-polyisoprene copolymer, a neoprene, a nitrile rubber, a polyisobutylene, an olefinic rubber, an ethylene-propylene rubber, an ethylene-propylene-diene monomer rubber, a polyurethane elastomer, a silicone rubber, a fluoroelastomer, a fluorosilicone rubber, a vinyl acetate homopolymer, a vinyl acetate copolymer, an ethylene vinyl acetate copolymer, an acrylates homopolymer, an acrylates copolymer, polymethylmethacrylate, polyethylmethacrylate, polymethacrylate, ethylene glycol dimethacrylate, ethylene dimethacrylate, hydroxymethyl methacrylate, a polyvinylpyrrolidone, a polyacrylonitrile butadiene, a polycarbonate, a polyamide, a fluoropolymer, polytetrafluoroethylene, polyvinyl fluoride, a polystyrene, a styrene acrylonitrile homopolymers, a styrene acrylonitrile copolymer, a cellulose acetate, an acrylonitrile butadiene styrene homopolymer, a acrylonitrile butadiene styrene copolymer, a polymethylpentene, a polysulfone, a polyester, a polyimide, a polyisobutylene, a polymethylstyrene, an alginate, an agarose, a dextrin, a dextran, a multiblock polymer, a biocompatible metal alloy, titanium, platinum, stainless steel, hydroxyapatite, tricalcium phosphate, cocoa butter, a wax, and a ceramic material. Preferably, the biodegradable polymer is a polylactate/polyglycolate copolymer.
The invention also relates to a surface coated with the composition of the invention.
The invention further relates to an implantable device having a surface coated with the composition of the invention. The device may, for example, be selected from the group consisting of a wound dressing, a suture, a particle, a vascular stent, and a bulk material.
When the device is a vascular stent, the biodegradable matrix is preferably a polylactate/polyglycolate copolymer, and the polyionic bioactive agent is preferably a nucleic acid selected from the group consisting of an expression vector encoding an anti-restenotic protein and an anti-restenotic antisense oligonucleotide, and wherein the exterior portion further comprises polylysine. The anti-restenotic protein may, for example, be selected from the group consisting of TPA, TGF-xcex2, FGF, Rb, p21, and TK. The anti-restenotic antisense oligonucleotide may, for example, be selected from the group consisting of a c-myb antisense oligonucleotide, a c-myc antisense oligonucleotide, and a PCNA antisense oligonucleotide.
When the device is a suture coated with a plurality of layers of the matrix, the biodegradable matrix is preferably a polylactate/polyglycolate copolymer, and the polyionic bioactive agent is preferably a nucleic acid expression vector encoding a wound healing therapeutic protein. The wound healing therapeutic protein may, for example, be selected from the group consisting of TGF-xcex2, FGF, PDGF, IGF, M-CGF, BMP, GH, and PTH. Preferably, suture is coated with at least twenty layers of the matrix.
When the device is a particle, the polyanionic bioactive agent is preferably selected from the group consisting of an expression vector encoding a wound healing therapeutic protein, an expression vector encoding an anti-restenotic protein, and an anti-restenotic antisense oligonucleotide. The wound healing therapeutic protein may, for example, be selected from the group consisting of TGF-xcex2, FGF, PDGF, IGF, M-CGF, BMP, GH, and PTH. The anti-restenotic protein may, for example, be selected from the group consisting of TPA, TGF-xcex2, FGF, Rb, p21, and TK. The anti-restenotic antisense oligonucleotide is selected from the group consisting of a c-myb antisense oligonucleotide, a c-myc antisense oligonucleotide, and a PCNA antisense oligonucleotide. Preferably, the particle has a diameter no greater than about 900 micrometers, and more preferably no greater than about 1 micrometer.
When the device is a bulk material, the polyanionic bioactive agent is preferably selected from the group consisting of an expression vector encoding an oncogene and an antisense oligonucleotide directed against an oncogene. The oncogene may, for example, be selected from the group consisting of abl, akt2, apc, bcl2xcex1, bcl2xcex2, bcl3, bcr, brcal, brca2, cbl, ccnd1, cdk4, crk-II, csf1r/fms, dbl, dcc, dpc4/smad4, e-cad, e2f1/rbap, egfr/erbb-1, elk1, elk3, eph, erg, ets1, ets2, fer, fgr/src2, fli1/ergb2, fos, fps/fes, fra1, fra2, fyn, hck, hek, her2/erbb-2/neu, her3/erbb-3, her4/erbb-4, hras1, hst2, hstf1, ink4a, ink4b, int2/fgf3, jun, junb, jund, kip2, kit, kras2a, kras2b, lck, lyn, mas, max, mcc, met, mlh1, mos, msh2, msh3, msh6, myb, myba, mybb, myc, mycl1, mycn, nf1, nf2, nras, p53, pdgfb, pim1, pms1, pms2, ptc, pten, raf1, rb1, rel, ret, ros1, ski, src1, tal1, tgfbr2, thra1, thrb, tiam1, trk, vav, vhl, waf1, wnt1, wnt2, wt1, and yes1.
When a surface is coated with the composition of the invention having an interior portion having the polyionic bioactive agent suspended therein, the interior portion is preferably interposed between the surface and the exterior portion of the matrix. Thus, for example, when an implantable device has a surface coated with the composition having such an interior portion, the interior portion is preferably interposed between the surface of the device and the exterior portion of the matrix.
The invention also relates to a method of making a composition for delivery of a polyionic bioactive agent. This method comprises providing a biodegradable matrix having an interior portion and an exterior portion and contacting the exterior portion of the biodegradable matrix with a polyionic condensing agent having a charge opposite that of the polyionic bioactive agent. The polyionic bioactive agent is suspended in the biodegradable matrix in a non-condensed form. At least most of the polyionic bioactive agent assumes a condensed form at the exterior portion of the biodegradable matrix when it is contacted with the polyionic condensing agent.
The invention further relates to a method of making a composition for delivery of a polyionic bioactive agent. This method comprises providing a matrix which has an exterior portion and which comprises the polyionic bioactive agent at the exterior portion; and contacting the exterior portion of the matrix with a polyionic condensing agent having a charge opposite that of the polyionic bioactive agent. At least most the polyionic bioactive agent assumes a condensed form at the exterior portion of the matrix.
The invention still further relates to a method of delivering a polyionic bioactive agent to an animal tissue. This method comprising placing in fluid communication with the animal tissue a composition comprising the polyionic bioactive agent and a matrix having an exterior portion. At least most of the polyionic bioactive agent is in a condensed form at the exterior portion of the matrix.
The invention also relates to a kit comprising a biocompatible matrix having an exterior portion and an instructional material which describes combining the matrix with a polyionic bioactive agent and condensing at least most of the polyionic bioactive agent at the exterior portion of the matrix.
The invention relates to another kit, this kit comprising a composition which comprises the polyionic bioactive agent and a biocompatible matrix having an exterior portion. At least most of the polyionic bioactive agent present at the exterior portion of the matrix is in a condensed form. The kit further comprises an instructional material which describes administration of the composition to a tissue of an animal to effect delivery of the polyionic bioactive agent to the tissue.
In another aspect, the invention relates to a kit for coating an implantable device with a composition for delivery of a polyionic bioactive agent upon implantation of the device. This kit comprises a biocompatible polymeric matrix suspended in a solvent and a polyionic condensing agent having a charge opposite that of the polyionic bioactive agent.
The invention also includes a kit for coating an implantable device with a composition for delivery of a polyionic bioactive agent upon implantation of the device. This kit comprising a suspension of monomers of a biocompatible polymeric matrix, a polymerization initiator, and a polyionic condensing agent having a charge opposite that of the polyionic bioactive agent.
The invention further includes a method of storing a nucleic acid. This storage method comprises suspending the nucleic acid in a matrix and contacting the matrix with a polycationic condensing agent.